Bottom Line:
TIMP-2 expression did not increase in U87MG cells.MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group.Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Background: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.

Methods: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.

Results: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Conclusion: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.

Fig3: Radiation effect on p53, MMP-2 and TIMP-2 in U87 MG-Fluc cells. Establishment bioluminescent tumor cells “U87-Fluc” which is transfected with firefly luciferase (fluc) in u87MG. a Photons emitted from U87-Fluc cell line when D-luciferin was added; b Western blot; c Zymography for detection MMP-2 activity in dependent on time; d Density of MMP-2 activity. It had been showed statistical significance between control and 2 Gy IR from 30 hr (p < 0.05)

Mentions:
We established the bioluminescent tumor cell line “U87-Fluc” for in vivo experiments, in which u87MG was transfected with firefly luciferase (Fluc). This is a useful tool to observe tumor formation without sacrifice after implanting tumor cells in the mouse brain. The U87-Fluc cell line emitted photons when D-luciferin was added (Fig. 3a), and U87-Fluc cells increased p53 expression and MMP-2 activity in proportion to the radiation dose but did not affect TIMP-2 expression (Fig. 3b, c). MMP-2 activity increased depending on the incubation time after radiation exposure and showed a great difference between control and radiated cells after 24 hr (p < 0.05, Fig. 3d).Fig. 3

Fig3: Radiation effect on p53, MMP-2 and TIMP-2 in U87 MG-Fluc cells. Establishment bioluminescent tumor cells “U87-Fluc” which is transfected with firefly luciferase (fluc) in u87MG. a Photons emitted from U87-Fluc cell line when D-luciferin was added; b Western blot; c Zymography for detection MMP-2 activity in dependent on time; d Density of MMP-2 activity. It had been showed statistical significance between control and 2 Gy IR from 30 hr (p < 0.05)

Mentions:
We established the bioluminescent tumor cell line “U87-Fluc” for in vivo experiments, in which u87MG was transfected with firefly luciferase (Fluc). This is a useful tool to observe tumor formation without sacrifice after implanting tumor cells in the mouse brain. The U87-Fluc cell line emitted photons when D-luciferin was added (Fig. 3a), and U87-Fluc cells increased p53 expression and MMP-2 activity in proportion to the radiation dose but did not affect TIMP-2 expression (Fig. 3b, c). MMP-2 activity increased depending on the incubation time after radiation exposure and showed a great difference between control and radiated cells after 24 hr (p < 0.05, Fig. 3d).Fig. 3

Bottom Line:
TIMP-2 expression did not increase in U87MG cells.MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group.Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Background: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.

Methods: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.

Results: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Conclusion: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.